The aim of the Molecular Therapeutics Unit (MTU) is the identification of novel biochemical pathways altered in HNSCC leading to the discovery of small molecules that may modulate molecular events important for oral carcinogenesis, and thus may represent novel chemotherapeutic agents. Furthermore, the unit focuses in the preclinical assessment of biochemical and molecular parameters modulated by these small molecules, which may help to monitor the effects on tumor samples in patients with head and neck cancer receiving these novel treatment modalities. Finally, selected signaling agents developed in the unit are tested in early clinical trials in patients with advanced HNSCC and other malignancies for ?proof of principle testing?, in order to assess whether the preclinical effects observed occur also in humans; novel effects observed in human trials are then, investigated in our unit, ?reverse translation?. 1)Mechanism of antiproliferative effects of small molecules cell cycle modulators Perifosine (per) is a novel alkylphospholipid with antitumor properties, derived from miltefosine, an approved drug in Europe for the treatment of cutaneous malignancies. The exact mechanism of action is still unknown. We recently established that perifosine promotes cell cycle arrest by increase in the endogenous cdk inhibitor p21waf1 protein levels. To discern the mechanism of p21 upregulation, northern blot studies revealed that the accumulation of p21 is transcriptional. The minimal p21 promoter region required for perifosine effects is identical to the region required for Ras to activate p21 promoter. This region contains several sp1 sites. Mutations in each specific Sp1 site abolished the activation by perifosine. Moreover, we demonstrated that perifosine, similar to Ras, activates the MEK/ERK pathway. Furthermore, perifosine is able to phosphorylate Sp1 in MAPK-dependent sites, and this phosphorylation is required for Sp1 increased DNA binding (as measured by gel shift analysis). Finally, the transcriptional induction of p21 by perifosine requires MEK/ERK activation as chemical or genetic inhibitors of MEK, (PD98059 or MEKAA, respectively) abolished p21 transactivation, p21 induction and increase Sp1 DNA binging activity, concluding that Per activates the p21 promoter via activation of MEK by modulation of Sp1. The small molecule, UCN-01, is a cdk modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models, currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear. We have previously reported that UCN-01 promotes G1/S cell cycle arrest in a battery of head and neck squamous cancer cell lines (HNSCC). The arrest is accompanied by an increase in both p21cip1/waf1 and p27kip1 cdk inhibitors leading to loss in G1 cdk activity. To determine whether p21 or p27 are required for UCN-01 cell cycle effects, we tested HCT116 isogenic clones demonstrating that p21 was required for the cell cycle effects of UCN-01, as HCT116 lacking p21 (HCT 116 p21 -/-) were refractory to UCN-01?s cell cycle effects. Furthermore, we investigated the mechanism by which UCN-01 induces p21. UCN-01 promoted the accumulation of p21 at the mRNA level in the p53-deficient HaCaT cells without increase in the p21 mRNA half-life, suggesting that UCN-01 induced p21 at the transcriptional level. To study UCN-01 transcriptional activation of p21 we used several p21waf1/cip1 promoter-driven luciferase reporter plasmids and observed that UCN-01 activated the full-length p21waf1/cip1 promoter and a construct lacking p53 binding sites. The minimal promoter region required for UCN-01 (from -110 bp to the transcription start site) was the same minimal p21waff1/cip1 promoter region required for Ras enhancement of p21waf1/cip1 transcription. Neither PKC nor PDK1 /AKT pathways, known in vitro targets of UCN-01, were relevant for the induction of p21 by UCN-01. In contrast, the activation of MEK/ERK MAPK pathways was required for p21 induction as UCN-01 activated this pathway and genetic or chemical MEK inhibitors blunted p21 accumulation. These results demonstrated for the first time that p21waf1/cip1 is required for UCN-01 cell cycle arrest. Moreover, we showed that the accumulation of p21waf1/cip1 is transcriptional via activation of MEK pathway. This represents a novel mechanism by which UCN-01 exerts its antiproliferative effect. This promising mechanism should be exploited in future clinical trials using this agent.